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• W1992645522 abstract "Chromogranins A (CGA) and B (CGB) are two major Ca2+ storage proteins of the secretory granules of neuroendocrine cells. Nevertheless, we found in the present study that CGB was also localized in the nucleus. In immunogold electron microscopy using bovine adrenal medullary chromaffin cells, it was found that the number of CGB-labeled gold particles localized per μm2 of the nucleus was equivalent to 20% that of CGB-labeled gold particles localized per μm2 of the secretory granules. Considering that CGB is estimated to exist in the 0.1–0.2-mm range in the secretory granules of bovine chromaffin cells, 20% of these amounts to 20–40 μm. In addition, transfection of CGA and CGB into nonneuroendocrine COS-7 and NIH3T3 cells repeatedly indicated the nuclear localization of CGB in addition to its usual localization in the cytoplasm. Moreover, immunoblot and immunogold electron microscopy analyses of neuroendocrine PC12 cells also showed the existence of endogenous CGB in both the cytosol and the nucleus. Nuclear routing of CGB did not appear to depend entirely upon the nuclear localization signal as some of the nuclear localization signal mutant CGB were still targeted to the nucleus. In gene array assay, CGB was shown to either induce or suppress transcription of many genes including those of transcription factors. Of these we have analyzed eight genes, four induced (zinc finger protein, MEF2C, hCRP2, abLIM) and four suppressed (hcKrox, T3-receptor, troponin C, integrin) using the quantitative reverse transcription-PCR method and spectrophotometry to determine the transcription levels of each mRNA. CGB was shown to increase the transcription of zinc finger protein, MEF2C, hCRP2, and abLIM by 2.5–5-fold while suppressing that of hcKrox, T3-receptor, troponin C, and integrin by 60–75%. Given that MEF2C and hcKrox genes are transcription factors, these results pointed to the transcription control role of CGB in the nucleus. Chromogranins A (CGA) and B (CGB) are two major Ca2+ storage proteins of the secretory granules of neuroendocrine cells. Nevertheless, we found in the present study that CGB was also localized in the nucleus. In immunogold electron microscopy using bovine adrenal medullary chromaffin cells, it was found that the number of CGB-labeled gold particles localized per μm2 of the nucleus was equivalent to 20% that of CGB-labeled gold particles localized per μm2 of the secretory granules. Considering that CGB is estimated to exist in the 0.1–0.2-mm range in the secretory granules of bovine chromaffin cells, 20% of these amounts to 20–40 μm. In addition, transfection of CGA and CGB into nonneuroendocrine COS-7 and NIH3T3 cells repeatedly indicated the nuclear localization of CGB in addition to its usual localization in the cytoplasm. Moreover, immunoblot and immunogold electron microscopy analyses of neuroendocrine PC12 cells also showed the existence of endogenous CGB in both the cytosol and the nucleus. Nuclear routing of CGB did not appear to depend entirely upon the nuclear localization signal as some of the nuclear localization signal mutant CGB were still targeted to the nucleus. In gene array assay, CGB was shown to either induce or suppress transcription of many genes including those of transcription factors. Of these we have analyzed eight genes, four induced (zinc finger protein, MEF2C, hCRP2, abLIM) and four suppressed (hcKrox, T3-receptor, troponin C, integrin) using the quantitative reverse transcription-PCR method and spectrophotometry to determine the transcription levels of each mRNA. CGB was shown to increase the transcription of zinc finger protein, MEF2C, hCRP2, and abLIM by 2.5–5-fold while suppressing that of hcKrox, T3-receptor, troponin C, and integrin by 60–75%. Given that MEF2C and hcKrox genes are transcription factors, these results pointed to the transcription control role of CGB in the nucleus. chromogranin A chromogranin B inositol 1,4,5-trisphosphate receptor green fluorescent protein endoplasmic reticulum hemagglutinin phosphate-buffered saline electron microscope nuclear localization signal The secretory granules of neuroendocrine cells are loaded with hormones, neurotransmitters, and ions such as Ca2+, Mg2+, and Zn2+ along with peptides and proteins of which chromogranins A and B are the most abundant (1Winkler H. Westhead E. Neuroscience. 1980; 5: 1803-1823Crossref PubMed Scopus (370) Google Scholar, 2Simon J.-P. Aunis D. Biochem. J. 1989; 262: 1-13Crossref PubMed Scopus (242) Google Scholar, 3Helle K.B. Neurochem. Int. 1990; 17: 165-175Crossref PubMed Scopus (58) Google Scholar, 4Winkler H. Fischer-Colbrie R. Neuroscience. 1992; 49: 497-528Crossref PubMed Scopus (611) Google Scholar, 5Iacangelo A. Eiden L.E. Regul. Pept. 1995; 58: 65-88Crossref PubMed Scopus (154) Google Scholar). Chromogranins A and B are acidic proteins (1Winkler H. Westhead E. Neuroscience. 1980; 5: 1803-1823Crossref PubMed Scopus (370) Google Scholar, 2Simon J.-P. Aunis D. Biochem. J. 1989; 262: 1-13Crossref PubMed Scopus (242) Google Scholar, 3Helle K.B. Neurochem. Int. 1990; 17: 165-175Crossref PubMed Scopus (58) Google Scholar, 4Winkler H. Fischer-Colbrie R. Neuroscience. 1992; 49: 497-528Crossref PubMed Scopus (611) Google Scholar, 5Iacangelo A. Eiden L.E. Regul. Pept. 1995; 58: 65-88Crossref PubMed Scopus (154) Google Scholar) with acidic residues constituting 25–30% of the amino acid residues (6Iacangelo A. Affolter H.-U. Eiden L.E. Herbert E. Grimes M. Nature. 1986; 323: 82-86Crossref PubMed Scopus (258) Google Scholar, 7Benedum U.M. Baeuerle P.A. Konecki D.S. Frank R. Powell J. Mallet J. Huttner W.B. EMBO J. 1986; 5: 1495-1502Crossref PubMed Scopus (204) Google Scholar, 8Ahn T.G. Cohn D.V. Gorr S.V. Ornstein D.L. Kashdan M.A. Levine M.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5043-5047Crossref PubMed Scopus (35) Google Scholar, 9Bauer J.W. Fischer-Colbrie R. Biochim. Biophys. Acta. 1991; 1089: 124-126Crossref PubMed Scopus (26) Google Scholar, 10Benedum U.M. Lamouroux A. Konecki D.S. Hille A. Baeuerle P.A. Frank R. Lottspeich F. Mallet J. Huttner W.B. EMBO J. 1997; 6: 1203-1211Crossref Scopus (191) Google Scholar, 11Pohl T.M. Phillips E. Song K. Gerdes H.-H. Hutter W.B. Rüther U. FEBS Lett. 1990; 262: 219-224Crossref PubMed Scopus (59) Google Scholar, 12Yoo S.H. Kang Y.K. FEBS Lett. 1997; 406: 259-262Crossref PubMed Scopus (24) Google Scholar), and this high content of negatively charged amino acid residues is thought to be responsible for the high capacity, low affinity Ca2+binding property of chromogranins (13Bulenda D. Gratzl M. Biochemistry. 1985; 24: 7760-7765Crossref PubMed Scopus (74) Google Scholar, 14Yoo S.H. Albanesi J.P. J. Biol. Chem. 1991; 266: 7740-7745Abstract Full Text PDF PubMed Google Scholar), binding 32–93 mol of Ca2+/mol (14Yoo S.H. Albanesi J.P. J. Biol. Chem. 1991; 266: 7740-7745Abstract Full Text PDF PubMed Google Scholar, 15Oh S.H. Yoo Y.S. Kang M.K. So Y.H. Huh S.H. Park H.S. Park H.Y. J. Biol. Chem. 2001; 276: 45806-45812Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar).The comparison of the amino acid sequences of CGA1 (6Iacangelo A. Affolter H.-U. Eiden L.E. Herbert E. Grimes M. Nature. 1986; 323: 82-86Crossref PubMed Scopus (258) Google Scholar, 7Benedum U.M. Baeuerle P.A. Konecki D.S. Frank R. Powell J. Mallet J. Huttner W.B. EMBO J. 1986; 5: 1495-1502Crossref PubMed Scopus (204) Google Scholar, 8Ahn T.G. Cohn D.V. Gorr S.V. Ornstein D.L. Kashdan M.A. Levine M.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5043-5047Crossref PubMed Scopus (35) Google Scholar) and CGB (9Bauer J.W. Fischer-Colbrie R. Biochim. Biophys. Acta. 1991; 1089: 124-126Crossref PubMed Scopus (26) Google Scholar, 10Benedum U.M. Lamouroux A. Konecki D.S. Hille A. Baeuerle P.A. Frank R. Lottspeich F. Mallet J. Huttner W.B. EMBO J. 1997; 6: 1203-1211Crossref Scopus (191) Google Scholar, 11Pohl T.M. Phillips E. Song K. Gerdes H.-H. Hutter W.B. Rüther U. FEBS Lett. 1990; 262: 219-224Crossref PubMed Scopus (59) Google Scholar, 12Yoo S.H. Kang Y.K. FEBS Lett. 1997; 406: 259-262Crossref PubMed Scopus (24) Google Scholar) shows little sequence homology except the two conserved regions, one near the N-terminal region bordered by two cysteine residues (residues 17–38 in bovine CGA and 16–37 in bovine CGB) and the other the C-terminal region (residues 409–431 in bovine CGA and 604–626 in bovine CGB). Despite the differences in amino acid sequences, chromogranins A and B and secretogranin II (also called chromogranin C) were shown to aggregate in an acidic pH and high calcium environment (16Gerdes H.-H. Rosa P. Phillips E. Baeuerle P.A. Frank R. Argos P. Huttner W.B. J. Biol. Chem. 1989; 264: 12009-12015Abstract Full Text PDF PubMed Google Scholar, 17Gorr S.-U. Shioi J. Cohn D.V. Am. J. Physiol. 1989; 257: E247-E254PubMed Google Scholar, 18Yoo S.H. Albanesi J.P. J. Biol. Chem. 1990; 265: 14414-14421Abstract Full Text PDF PubMed Google Scholar, 19Chanat E. Huttner W.B. J. Cell Biol. 1991; 115: 1505-1519Crossref PubMed Scopus (386) Google Scholar, 20Yoo S.H. J. Biol. Chem. 1995; 270: 12578-12583Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), the condition found in the trans-Golgi network. Nevertheless, there was a big difference in the pH- and Ca2+-dependent aggregation properties of these two proteins; the aggregation of CGB being at least two orders of magnitude more sensitive to Ca2+ than CGA (20Yoo S.H. J. Biol. Chem. 1995; 270: 12578-12583Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Moreover, unlike CGA, which dimerized at pH 7.5 and tetramerized at pH 5.5 (21Yoo S.H. Lewis M.S. J. Biol. Chem. 1992; 267: 11236-11241Abstract Full Text PDF PubMed Google Scholar,22Thiele C. Huttner W.B. J. Biol. Chem. 1998; 273: 1223-1231Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), purified CGB appeared to exist in a monomeric state (20Yoo S.H. J. Biol. Chem. 1995; 270: 12578-12583Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar).We have shown previously that CGA and CGB, as well as most of the secretory vesicle matrix proteins, not only aggregated in the presence of Ca2+ at the intravesicular pH 5.5 but also bound to several integral membrane proteins of the secretory granule, including the IP3R (23Yoo S.H. J. Biol. Chem. 1994; 269: 12001-12006Abstract Full Text PDF PubMed Google Scholar, 24So S.H. Yoo S.H. Kweon H.S. Lee J.S. Kang M.K. Jeon C.J. J. Biol. Chem. 2000; 275: 12553-12559Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Some of the vesicle matrix proteins that failed to bind to the vesicle membrane were shown to bind instead to CGA, thus ensuring their interaction with the vesicle membrane (25Yoo S.H. J. Biol. Chem. 1996; 271: 1558-1565Abstract Full Text Full Text PDF PubMed Google Scholar). Hence, in view of the chromogranins' ability to interact with both the vesicle matrix proteins and the vesicle membrane, the roles of CGA and CGB in the selective aggregation and the sorting of potential vesicle matrix proteins to the secretory granules appear to be essential in secretory granule biogenesis (25Yoo S.H. J. Biol. Chem. 1996; 271: 1558-1565Abstract Full Text Full Text PDF PubMed Google Scholar, 26Huttner W.B. Gerdes H.-H. Rosa P. Trends Biochem. Sci. 1991; 16: 27-30Abstract Full Text PDF PubMed Scopus (415) Google Scholar). Thus, chromogranins A and B have been suggested to play key roles in secretory granule biogenesis (5Iacangelo A. Eiden L.E. Regul. Pept. 1995; 58: 65-88Crossref PubMed Scopus (154) Google Scholar,25Yoo S.H. J. Biol. Chem. 1996; 271: 1558-1565Abstract Full Text Full Text PDF PubMed Google Scholar, 26Huttner W.B. Gerdes H.-H. Rosa P. Trends Biochem. Sci. 1991; 16: 27-30Abstract Full Text PDF PubMed Scopus (415) Google Scholar). It was indeed reported recently that CGA functions as an on/off switch for secretory granule biogenesis in PC12 cells (27Kim T. Tao-Cheng J.-H. Eiden L.E. Loh Y.P. Cell. 2001; 106: 499-509Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). Using the antisense RNA technique and PC12 cells, Kim et al.(27Kim T. Tao-Cheng J.-H. Eiden L.E. Loh Y.P. Cell. 2001; 106: 499-509Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar) showed that the number of secretory granules formed is directly related to the amount of CGA expressed in PC12 cells. It was further shown that the secretory granule formation could be induced in nonneuroendocrine cells, which normally don't contain any secretory granules, by expressing CGA in these cells.We have extended here the chromogranin study and found that CGB was also localized in the nucleus in addition to its usual presence in the secretory granules. Although other secretory granule resident proteins proenkephalin and corticotrophin-releasing hormone have also been found in the nucleus before (28Böttger A. Spruce B.A. J. Cell Biol. 1995; 130: 1251-1262Crossref PubMed Scopus (34) Google Scholar, 29Castro M.G. Morrison E. Tomasec P. Linton E.A. Lowenstein P.R. Cell Tissue Res. 1995; 282: 367-376Crossref PubMed Scopus (4) Google Scholar), this is the first time the secretory granule marker protein chromogranin is found in the nucleus, opening new possibilities for the role of chromogranin in the nucleus. One of the nuclear roles of CGB appears to be control of the transcription of many genes, including those for transcription factors.RESULTSIn view of the abundant presence of CGA and CGB in the adrenal chromaffin cells, we investigated the possibility of the presence of endogenous CGB in the nucleus of bovine adrenal medullary chromaffin cells using immunogold electron microscopy (Fig.1). As shown in Fig. 1A, the CGA-labeled gold particles were primarily localized in the secretory granules with some in the endoplasmic reticulum. But virtually no CGA-labeling gold particles were found in the mitochondria. In contrast, the CGB-labeled gold particles localized not only in the secretory granules but also in the nucleus (Fig. 1B). Like the result in Fig. 1A, the chromogranin B-labeled gold particles were not found in the mitochondria. In control experiments, omission of the primary antibody or preimmune treatment in place of the primary antibody almost completely eliminated the chromogranin-labeled gold particles (Fig. 1C)To further evaluate the relative abundance of CGB in the nucleus, we examined fifteen different EM images, which had been prepared from seven different tissue samples and counted the total number of CGB-labeled gold particles in the secretory granules, nucleus, and mitochondria (Table I). As shown in TableI, 1027 CGB-labeled gold particles were found in 6.42 μm2of the secretory granule area thus averaging 160 CGB-labeled gold particles per μm2 of the secretory granule area. In the same EM images 636 CGB-labeled gold particles were found in 19.47 μm2 of the nuclear area averaging 33 CGB-labeled gold particles per μm2, whereas 12 gold particles were localized in 3.51 μm2 of mitochondria averaging three gold particles per μm2. In light of the fact that two-three gold particles were consistently found per μm2of adrenal chromaffin cells in the control EM images, the three CGB-labeled gold particles found per μm2 of mitochondria are considered to result from nonspecific interactions.Table IDistribution of the CGA- and CGB-labeled gold particles in bovine adrenal medullary chromaffin cellCGACGBNumber of gold particles1-a15 images from five different tissue preparations were used./area viewed (μm2)Gold particles/μm2Number of gold particles1-b15 images from seven different tissue preparations were used./area viewed (μm2)Gold particles/μm2Secretory granule3488 /7.984371027 /6.42160Nucleus40 /13.773636 /19.4733Mitochondria13 /5.28212 /3.5131-a 15 images from five different tissue preparations were used.1-b 15 images from seven different tissue preparations were used. Open table in a new tab Similar to the CGB-immunogold study, fifteen different images from five different tissue samples were also examined for the presence of CGA-labeled gold particles in the secretory granules, nucleus, and mitochondria (Table I). The CGA-labeled gold particles were found virtually in all the secretory granules, averaging 437 CGA-labeled gold particles per μm2 of the secretory granule, whereas 2–3 gold particles each were found per μm2 of the nucleus and of the mitochondria. Again, the two-three gold particles that were found per μm2 of the nucleus or mitochondria are identical to the number of gold particles found in the absence of the primary antibody. This result is in contrast to that obtained with the CGB-labeled gold particles, which clearly demonstrated the presence of CGB in the nucleus.To determine whether transfected CGB can be routed to the nucleus in nonneuroendocrine cells, we have constructed CGA and CGB expression vectors and transfected them into COS-7 cells. The bovine chromogranins A and B that were used in the present experiments are shown in Fig.2A. Two conserved regions (near the N-terminal and the C-terminal regions) are indicated asdashed boxes in Fig. 2A. For the immunolabeling and immunoblotting procedures, we tagged CGA and CGB at the C-terminal ends either with HA and His6, respectively, or with GFP. When chromogranins A and B were introduced into COS-7 cells, the expression of CGA and CGB in the cells was confirmed by immunoblot analysis using either the tagging peptide-specific or chromogranin-specific antibody, which indicated a normal expression of the transfected chromogranins in COS-7 cells (Fig. 2B). In the cells that had been transfected with the control vector (pCI-neo), no band was present.Figure 2Construction and expression of bovine CGA and CGB in transiently transfected COS-7 cells. A, schematic diagrams for bovine CGA and CGB used in the transfection analysis. For differential expression, CGA and CGB were tagged with HA (CGA-HA) and His6 (CGB-His) at the C-terminal ends, respectively. The location of highly conserved regions is indicated as gray boxes. Solid boxes at the N-terminal ends of CGA and CGB indicate the leader sequence (L). B, Western analysis of CGA and CGB in the COS-7 cell extracts. The total protein extracts resolved on an 8% SDS-gel were probed serially with the anti-HA and anti-CGA antibodies for CGA (left two panels) and with the anti-His and anti-CGB antibodies for CGB (right two panels). The extract from the pCI-neomycin vector-transfected cells was used as a control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The nuclear localization of CGB was also evident when the CGB-GFP fusion protein was expressed in COS-7 cells (Fig.3C). Transfection of the cells with GFP only indicated the expression of GFP throughout the cell with a bit brighter fluorescence in the nuclear area (Fig. 3A). The diffuse fluorescence indicates that GFP is localized both in the cytoplasm and the nucleus, and the brighter fluorescence in the nuclear area probably reflects a greater depth of view of the nuclear area when viewed with a fluorescence microscope. But the CGA-GFP expression was limited to punctate localization in the cytoplasm with no localization in the nucleus (Fig. 3B). The punctate localization of CGA-GFP suggests granular localization of CGA-GFP. Though nonneuroendocrine COS-7 cells do not contain secretory granules, it appears apparent that granular structures were found in the CGA-transfected cells, which is consistent with the published results that demonstrated the secretory granule formation in the CGA-transfected nonneuroendocrine cells (27Kim T. Tao-Cheng J.-H. Eiden L.E. Loh Y.P. Cell. 2001; 106: 499-509Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). In contrast, the CGB-GFP expression was evident in both the cytoplasm and the nucleus (Fig.3C). The cytoplasmic CGB-GFP fluorescence was shown in punctate structures, suggesting the localization of CGB-GFP in granular structures, whereas the nuclear fluorescence did not appear in punctate structures.Figure 3Localization of CGA-GFP and CGB-GFP in COS-7 cells. The C-terminally tagged chromogranin-GFP fusion proteins were expressed in COS-7 cells. The CTL-GFP (A) indicates transfection of GFP alone (control) while CGA-GFP (B) and CGB-GFP (C) indicate COS-7 cells transiently transfected with CGA- and CGB-GFP, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine the subcellular localization of transfected CGA and CGB in COS-7 and NIH3T3 cells, immunoblot analysis of the protein extracts of the chromogranin-transfected COS-7 and NIH3T3 cells was carried out (Fig. 4). As shown in Fig. 4A, CGB was detected in the nucleus of the CGB-transfected COS-7 cells, and its level was similar to that of the cytosol, but CGA was not detected in the nuclear extract of CGA-transfected COS-7 cells. Similarly, CGB was detected in the nuclear extract of the CGB-transfected NIH3T3 cells (Fig. 4B), but CGA was not detected in the nucleus of CGA-transfected NIH3T3 cells. The purity of the cytosolic and nuclear protein extracts was ensured by examining the existence of the ER marker protein calnexin in the cytosolic proteins and of the nucleus marker protein histone-4 in the nuclear proteins (Fig. 4, Aand B).Figure 4Localization of transfected CGA and CGB in COS-7 and NIH3T3 cells. The presence of CGA and CGB in the protein extracts of total (T), cytosolic (C), and nuclear (N) fractions of COS-7 cells transfected with CGA-GFP and CGB-GFP (A), respectively, and of NIH3T3 cells transfected with CGA-His and CGB-His (B), respectively, was analyzed by immunoblotting using the monoclonal CGA and CGB antibodies. Separation of the cytosolic and nuclear proteins was also ensured by immunoblotting with the polyclonal nuclear marker protein histone-4 and ER marker protein calnexin antibodies. 50 μg of the protein extract per lane was loaded for CGA and CGB immunoblots, but 10 μg of the proteins was loaded for the calnexin and histone-4 immunoblots.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether the nuclear routing of CGB is due to the overexpression of CGB in the CGB-transfected cells, the expression levels of transfected CGA and CGB were determined by measuring the expression levels of the GFP, which had been tagged to both CGA and CGB, in the CGA-GFP- and CGB-GFP-transfected cells (Fig.5A). As shown in Fig.5A, the CGB-GFP expression levels were only one-third or less those of CGA-GFP in NIH3T3 and COS-7 cells. This indicated that the amount of transfected CGB in the chromogranin-transfected NIH3T3 and COS-7 cells is one-third or less that of transfected CGA. Therefore, the nuclear localization of CGB cannot be due to the overexpression of CGB in these cells. Even after taking the larger molecular size of CGB (71 kDa) compared with that of CGA (48 kDa) into consideration, it is obvious that the nuclear routing of CGB is not the result of overexpression of CGB.Figure 5Expression levels of CGA-GFP and CGB-GFP and the separate localization of CGA and CGB in the cells cotransfected with CGA-HA and CGB-His. A, the presence of GFP in the total protein extracts (50 μg/lane) of NIH3T3 and COS-7 cells each transfected with CGA-GFP and CGB-GFP, respectively, was analyzed by immunoblotting using the monoclonal GFP antibody. B, the presence of CGA and CGB in the protein extracts (50 μg/lane) of total (T), cytosolic (C), and nuclear (N) fractions of COS-7 cells cotransfected with CGA-HA and CGB-His (CGA+ CGB) was analyzed using the HA- (CGA-HA) and His- (CGB-His) specific antibodies.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further examine whether the nuclear routing of CGB can still occur in the nonneuroendocrine cells transfected with both CGA and CGB, CGA-HA and CGB-His were cotransfected into COS-7 cells. The coexpression of CGA and CGB in the same cells was confirmed by labeling the expressed CGA with fluorescein isothiocyanate and CGB with tetramethylrhodamine isothiocyanate, respectively. The immunoblot analysis of the presence of CGA and CGB in the protein extracts of these cells showed the targeting of CGB both to the nucleus and to the cytoplasm and of CGA to the cytoplasm only (Fig. 5B). Identical results were also obtained with the CGA construct tagged with His and the CGB construct tagged with HA. This result indicated that a nuclear routing mechanism that carries CGB, not CGA, to the nucleus is in operation.In view of the fact that PC12 cells contain endogenous CGB (32Rosa P. Hille A. Lee R.W.H. Zanini A. de Camilli P. Huttner W.B. J. Cell Biol. 1985; 101: 1999-2011Crossref PubMed Scopus (306) Google Scholar), we explored the possibility of detecting endogenous CGB in the nucleus of neuroendocrine PC12 cells by immunoblot analysis (Fig.6). As shown in Fig. 6, endogenous CGB was detected in both the cytosol and the nucleus of these cells although the CGB level in the nucleus was approximately one-third to one-fourth that of cytoplasm. However, unlike CGB, which was detected both in the cytoplasm and in the nucleus of nontransfected PC12 cells, CGA was detected in the cytoplasm but not in the nucleus. The immunoblot analysis of the protein extracts from these cells with the antibodies for the nucleus marker protein histone-4 and the endoplasmic reticulum marker calnexin ensured the lack of cross-contamination of these protein extracts.Figure 6Presence of endogenous CGB in PC12 cells. The presence of endogenous CGA or CGB in the protein extracts of total (T), cytosolic (C), and nuclear (N) fractions of PC12 cells was analyzed by immunoblotting using the monoclonal CGA and CGB antibodies and the polyclonal nuclear marker protein histone-4 and ER marker protein calnexin antibodies. 50 μg of the protein per lane was loaded for the CGA and CGB immunoblots, but 10 μg of the proteins was loaded for the calnexin and histone-4 immunoblots.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The immunogold electron microscopy of PC12 cells also indicated the presence of CGB in the nucleus (Fig. 7). Fig. 7A shows the CGB-labeling gold particles in the secretory granules and the nucleus but not in the mitochondria. In the same experiments, omission of the primary antibody or treatment with the preimmune serum in place of the primary antibody eliminated the gold particles almost completely (Fig. 7B), indicating the specific nature of the CGB-labeling immunogold EM results.Figure 7Immunogold electron microscopy showing the localization of CGB in PC12 cells. PC12 cells were immunolabeled for CGB (10 nm gold) with the affinity-purified CGB antibody (A). The CGB-labeling gold particles are localized both in the secretory granules (SG) and in the nucleus (Nu). Some of the CGB-labeling gold particles are shown in the endoplasmic reticulum (rer) but not in the mitochondria (M). Either omission or replacement of the primary antibody with the preimmune serum eliminated virtually all the gold particles in the same PC12 cells (B). Bar = 200 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether there exists the nuclear localization signal (NLS) sequence in CGB we subjected the bovine CGB sequence to the PSORT II program (33Nakai K. Horton P. Trends Biochem. Sci. 1999; 24: 34-36Abstract Full Text Full Text PDF PubMed Scopus (1824) Google Scholar) and found that bovine CGB contains a putative NLS sequence (34Kalderon D. Roberts B.L. Richardson W.D. Smith A.E. Cell. 1984; 39: 499-509Abstract Full Text PDF PubMed Scopus (1854) Google Scholar), Pro-Glu-Val-Asp-Lys-Arg-Arg (PEVDKRR) starting at residue 235 (Fig. 8A). This type of NLS starts with Pro and followed by, within three residues, a basic segment containing three of four Lys/Arg residues (reviewed in Ref. 35Hicks G.R. Raikhel N.V. Annu. Rev. Cell Dev. Biol. 1995; 11: 155-188Crossref PubMed Scopus (259) Google Scholar). Thus we tested whether this conserved sequence was responsible for the nuclear localization of CGB by introducing substitution mutations into the putative NLS sequence (Fig. 8A). In this mutant, proline and the critical three basic residues were substituted to serine and hydrophobic residues, resulting in Ser-Glu-Val-Asp-Leu-Gln-Leu (SEVDLQL). The expression of the transfected NLS mutant in COS-7 cells was confirmed by immunoblot analysis (Fig. 8B), and the proteins from the whole cells, cytosol, and the nucleus were examined for the presence of CGB (Fig. 8C). Similar to that of wild type CGB, the NLS mutant also expressed CGB both in the cytoplasm and in the nucleus. However, the relative amount of CGB targeted to the nucleus appeared to be significantly smaller than that shown in the wild type. This result indicated that the putative NLS sequence is not exclusively responsible for the nuclear routing of CGB, thereby suggesting the operation of additional regions of CGB or of f" @default.
• W1992645522 created "2016-06-24" @default.
• W1992645522 creator A5008099169 @default.
• W1992645522 creator A5016458533 @default.
• W1992645522 creator A5056944627 @default.
• W1992645522 creator A5060698282 @default.
• W1992645522 creator A5074424556 @default.
• W1992645522 creator A5075086123 @default.
• W1992645522 date "2002-05-01" @default.
• W1992645522 modified "2023-09-26" @default.
• W1992645522 title "Localization of the Secretory Granule Marker Protein Chromogranin B in the Nucleus" @default.
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